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filetable <- read.table(file="/Users/shubhamagrawal/Documents/work/compressed/DIA/fileTable.txt", header = TRUE)
mae <- readExperimentDIA(fileTable = filetable,
annotation_col = c("treatment", "timepoint", "replicate",
"sampleType"))
mae <- readRDS("data/maeObjNEW.Rds")
mae
## A MultiAssayExperiment object of 2 listed
## experiments with user-defined names and respective classes.
## Containing an ExperimentList class object of length 2:
## [1] Phosphoproteome: SummarizedExperiment with 40517 rows and 138 columns
## [2] Proteome: SummarizedExperiment with 8518 rows and 79 columns
## Functionality:
## experiments() - obtain the ExperimentList instance
## colData() - the primary/phenotype DataFrame
## sampleMap() - the sample coordination DataFrame
## `$`, `[`, `[[` - extract colData columns, subset, or experiment
## *Format() - convert into a long or wide DataFrame
## assays() - convert ExperimentList to a SimpleList of matrices
## exportClass() - save data to flat files
se <- mae[["Phosphoproteome"]]
colData(se) <- colData(mae[, colnames(se)])
se
## class: SummarizedExperiment
## dim: 40517 138
## metadata(0):
## assays(1): Intensity
## rownames(40517): s1 s2 ... s40516 s40517
## rowData names(6): UniprotID Gene ... Sequence site
## colnames(138): FullProteome_1stCtrl_0min_rep2
## FullProteome_1stCtrl_0min_rep3 ... Phospho_HGF_40min_rep1
## Phospho_HGF_6h_rep1
## colData names(6): treatment timepoint ... sample sampleName
plotIntensity(se[, se$sampleType == "Phospho"], color = "replicate") + theme_classic() +
theme(
axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0, size = 7),
plot.title = element_text(hjust = 0.5, face = "bold")
)
newSE <- preprocessPhos(seData = se, transform = "log2",
normalize = TRUE, impute = "QRILC")
## Warning in fun(libname, pkgname): mzR has been built against a different Rcpp version (1.0.12)
## than is installed on your system (1.1.0). This might lead to errors
## when loading mzR. If you encounter such issues, please send a report,
## including the output of sessionInfo() to the Bioc support forum at
## https://support.bioconductor.org/. For details see also
## https://github.com/sneumann/mzR/wiki/mzR-Rcpp-compiler-linker-issue.
## Imputing along margin 2 (samples/columns).
newSE
## class: SummarizedExperiment
## dim: 13081 59
## metadata(0):
## assays(2): Intensity imputed
## rownames(13081): s1 s4 ... s40511 s40514
## rowData names(6): UniprotID Gene ... Sequence site
## colnames(59): Phospho_1stCtrl_0min_rep2 Phospho_1stCtrl_0min_rep3 ...
## Phospho_HGF_40min_rep1 Phospho_HGF_6h_rep1
## colData names(6): treatment timepoint ... sample sampleName
plotIntensity(newSE, color = "replicate") + theme_classic() +
theme(
axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0, size = 7),
plot.title = element_text(hjust = 0.5, face = "bold")
)
plotMissing(newSE) + theme_classic() +
theme(
axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0, size = 7),
plot.title = element_text(hjust = 0.5, face = "bold")
)
pca <- stats::prcomp(t(assays(newSE)[["imputed"]]), center = TRUE, scale. = TRUE)
p <- plotPCA(pca = pca, se = newSE,
color = "treatment",
shape = "replicate")
p$layers[[1]]$aes_params$size <- 3
p$layers[[1]]$aes_params$stroke <- 1.2
p + coord_equal() + theme(aspect.ratio = 1, legend.position = "right",
axis.text = element_text(color = "#000000")) +
guides(
shape = guide_legend(order = 1),
color = guide_legend(order = 2)
)
newSE <- preprocessPhos(seData = se, transform = "log2",
normalize = TRUE, impute = "QRILC",
removeOutlier = "rep1")
## Imputing along margin 2 (samples/columns).
newSE
## class: SummarizedExperiment
## dim: 17035 32
## metadata(0):
## assays(2): Intensity imputed
## rownames(17035): s1 s4 ... s40513 s40514
## rowData names(6): UniprotID Gene ... Sequence site
## colnames(32): Phospho_1stCtrl_0min_rep2 Phospho_1stCtrl_0min_rep3 ...
## Phospho_HGF_40min_rep2 Phospho_HGF_40min_rep3
## colData names(6): treatment timepoint ... sample sampleName
pca <- stats::prcomp(t(assays(newSE)[["imputed"]]),
center = TRUE, scale. = TRUE)
p <- plotPCA(pca = pca, se = newSE,
color = "treatment",
shape = "replicate")
p$layers[[1]]$aes_params$size <- 3
p$layers[[1]]$aes_params$stroke <- 1.2
p + coord_equal() + theme(aspect.ratio = 1, legend.position = "right",
axis.text = element_text(color = "#000000")) +
guides(
shape = guide_legend(order = 1),
color = guide_legend(order = 2)
)
p <- plotHeatmap(type = "Top variant", newSE, top = 30, annotationCol = c("replicate", "treatment"))
g <- p$gtable
# Find row names grob
row_names <- which(g$layout$name == "row_names")
g$grobs[[row_names]]$gp <- gpar(fontsize = 8, fontface = "bold")
# Same for columns
col_names <- which(g$layout$name == "col_names")
g$grobs[[col_names]]$gp <- gpar(fontsize = 10)
grid.newpage()
grid.draw(g)
dea <- performDifferentialExp(se = newSE,
assay = "imputed",
method = "limma",
condition = "treatment",
reference = "1stCtrl",
target = "EGF")
p <- ggplot(dea$resDE, aes(x = pvalue)) +
geom_histogram(fill = "grey", col = "blue", alpha=0.7) +
ggtitle("P value histogram") + theme_classic() +
theme(plot.title = element_text(hjust = 0.5))
p
## `stat_bin()` using `bins = 30`. Pick better value `binwidth`.
pFilter = 0.01
plotVolcano(dea$resDE, pFilter = 0.01, fcFilter = 1) + theme_classic() +
geom_hline(yintercept = -log10(as.numeric(pFilter)), color = "brown",
linetype = "dashed") +
annotate(x = 3.0, y = -log10(as.numeric(pFilter)) - 0.20,
label = paste0("P-value = ", as.numeric(pFilter)),
geom = "text", size = 3.5, color = "brown")
plotVolcanoDEA(dea$resDE, fcFilter = 1, pFilter = 0.01, usePadj = FALSE)
dea$resDE
## # A tibble: 17,035 × 11
## ID log2FC stat pvalue padj UniprotID Gene Position Residue Sequence
## <I<c> <dbl> <dbl> <dbl> <dbl> <chr> <chr> <chr> <chr> <chr>
## 1 s5284 2.32 27.7 1.64e-11 2.79e-7 P04920 SLC4… 243 S QERRRIG…
## 2 s176… -3.54 -25.0 4.83e-11 4.11e-7 Q3MIN7 RGL3 573 S PAGSPPA…
## 3 s252… 6.59 23.4 9.85e-11 4.95e-7 Q8N6T3 ARFG… 304 S KAEGPLD…
## 4 s129… 3.21 23.1 1.16e-10 4.95e-7 Q07889 SOS1 1134 S PHGPRSA…
## 5 s282… 6.12 21.5 2.52e-10 8.59e-7 Q969V6 MRTFA 156 S LLSATSA…
## 6 s129… 5.27 20.2 4.72e-10 1.34e-6 Q07889 SOS1 1137 S PRSASVS…
## 7 s215… 3.74 19.9 5.79e-10 1.36e-6 Q6ZUM4 ARHG… 84 S APPGPHP…
## 8 s3876 5.03 19.7 6.40e-10 1.36e-6 O94763 URI1 492 T EFVSPSL…
## 9 s309… 4.29 19.1 8.65e-10 1.64e-6 Q9BQL6 FERM… 179 S TASGSSV…
## 10 s317… 4.38 16.8 3.37e- 9 5.06e-6 Q9BY89 KIAA… 1574 S PPSSRLS…
## # ℹ 17,025 more rows
## # ℹ 1 more variable: site <chr>
intensityBoxPlot(se = dea$seSub, id = 's4971', symbol = "EGFR_S991")
set.seed(12345)
library(matrixStats)
newSEts <- newSE[ , newSE$treatment == "EGF"]
assayMat <- SummarizedExperiment::assay(newSEts)
exprMat <- lapply(unique(newSEts$timepoint), function(tp) {
rowMedians(assayMat[,newSEts$timepoint == tp])
}) %>% bind_cols() %>% as.matrix()
## New names:
## • `` -> `...1`
## • `` -> `...2`
## • `` -> `...3`
## • `` -> `...4`
rownames(exprMat) <- rownames(newSEts)
colnames(exprMat) <- unique(newSEts$timepoint)
sds <- apply(exprMat,1,sd)
varPer <- 80
exprMat <- exprMat[order(sds, decreasing = TRUE)[seq(1, varPer/100*nrow(exprMat))], ]
# only center when it's for expression
exprMat <- mscale(exprMat)
# remove NA values
exprMat <- exprMat[complete.cases(exprMat), ]
tsc <- clusterTS(x = exprMat, k = 5, pCut = 0.6)
tsc$plot + theme(
axis.text.x = element_text(size = 10), axis.text = element_text(color = "#000000"), )
genesetPath <- system.file("shiny-app/geneset", package = "SmartPhos")
inGMT1 <- piano::loadGSC(paste0(genesetPath, "/Cancer_Hallmark.gmt"),
type="gmt")
resTab <- enrichDifferential(dea = dea$resDE, type = "Pathway enrichment",
gsaMethod = "PAGE", geneSet = inGMT1,
statType = "stat", nPerm = 200, sigLevel = 0.05,
ifFDR = FALSE)
## Running gene set analysis:
## Checking arguments...done!
## Final gene/gene-set association: 1218 genes and 50 gene sets
## Details:
## Calculating gene set statistics from 1218 out of 4183 gene-level statistics
## Removed 3168 genes from GSC due to lack of matching gene statistics
## Removed 0 gene sets containing no genes after gene removal
## Removed additionally 0 gene sets not matching the size limits
## Loaded additional information for 50 gene sets
## Calculating gene set statistics...done!
## Calculating gene set significance...done!
## Adjusting for multiple testing...done!
resTab
## Name Gene Number Stat p.up p.up.adj
## 1 HALLMARK_MITOTIC_SPINDLE 145 2.7227 0.0032376 0.16188
## 2 HALLMARK_HEDGEHOG_SIGNALING 10 -1.6913 0.9546100 0.99605
## 3 HALLMARK_MYC_TARGETS_V1 112 -1.9882 0.9766100 0.99605
## 4 HALLMARK_OXIDATIVE_PHOSPHORYLATION 23 -1.9984 0.9771600 0.99605
## 5 HALLMARK_KRAS_SIGNALING_DN 14 -2.6567 0.9960500 0.99605
## p.down p.down.adj Number up Number down
## 1 0.9967600 0.99676 90 55
## 2 0.0453920 0.45440 3 7
## 3 0.0233930 0.38989 60 52
## 4 0.0228360 0.38989 8 15
## 5 0.0039458 0.19729 4 10
inGMT2 <- piano::loadGSC(paste0(genesetPath, "/KEGG_pathways.gmt"),
type="gmt")
clustEnr <- clusterEnrich(clusterTab = tsc$cluster, se = newSE,
inputSet = inGMT2, filterP = 0.01, ifFDR = FALSE)
clustEnr$plot + theme_classic()
First on the differential expression analysis results
netw <- getDecouplerNetwork("Homo sapiens")
scoreTab <- calcKinaseScore(dea$resDE, netw, statType = "stat", nPerm = 100)
plotKinaseDE(scoreTab, nTop = 10, pCut = 0.05)
clusterData <- tsc$cluster[tsc$cluster$cluster == "cluster1",]
allClusterFeature <- clusterData %>%
distinct(feature, .keep_all = TRUE) %>% .$feature
allClusterSite <- data.frame(rowData(newSEts)[allClusterFeature, "site"])
allClusterSite$feature <- allClusterFeature
clusterData <- clusterData %>%
left_join(allClusterSite, by = "feature") %>%
rename(site = "rowData.newSEts..allClusterFeature...site..")
scoreTab <- calcKinaseScore(clusterData, netw, statType = "stat", nPerm = 100)
plotKinaseTimeSeries(scoreTab, pCut = 0.05, clusterName = "cluster1")
newSEts <- addZeroTime(newSE, condition = "treatment", treat = "EGF",
zeroTreat = "1stCtrl",
timeRange = c("20min","40min","100min"))
rd <- as.data.frame(rowData(newSEts))
timerange <- unique(newSEts$timepoint)
sites <- c("MET_Y1234", "MET_Y1235", "MET_1003", "Shc1_Y427", "AKT3_Y312", "RPS6KB1", "EGFR_Y1092", "EGFR_Y1172", "SOS1_S1134", "MAP2K2_S226", "MAPK3_T202", "MAPK1_T190", "EGFR_Y1197", "ERBB2_S998","GAB1_Y659","RPS6_S244")
for (i in sites) {
# condition to check if that site is present in row data
if (i %in% rd$site) {
# condition to check if assay doesn't have NAs (imputed assays are not allowed)
if (!is.na(assay(newSEts)[rownames(rd[rd$site==i,]), ][1])) {
p <- plotTimeSeries(newSEts, type = "expression",
geneID = rownames(rd[rd$site==i,]),
symbol = i, condition = "treatment",
treatment = "EGF", timerange = timerange) +
theme(axis.text = element_text(color = "#000000", size = 20),
axis.text.x = element_text(angle = 0, vjust = 0, hjust = 0.5, size = 20),
axis.title = element_text(size=20),
plot.title = element_text(size=30)) +
scale_x_continuous(breaks = c(0,20,40,60,80,100))
p$layers[[1]]$aes_params$size <- 6
p$layers[[1]]$mapping$colour <- NULL
p$layers[[1]]$aes_params$colour <- "#c1191f"
p$layers[[2]]$aes_params$linewidth <- 1
p$layers[[2]]$mapping$colour <- NULL
p$layers[[2]]$aes_params$colour <- "#555555"
print(p)
}
}
}